An increase of intracellular cAMP and cGMP produces potent inhibition of all platelet functions. Cyclic GMP inhibits cyclic AMP phosphodiesterase (cGI-PDE, PDE3A) and hydrolyzes cAMP, lowering its intracellular concentration. Inhibitors function as potent anti-platelet agents. Currently, the two anti-platelet agents with proven efficacy are aspirin, which inhibits cyclooxygenase-dependent synthesis of thromboxane A2 )TXA2), and clopidogrel or ticlopidine, which blocks away the ability of ADP to inhibit stimulated adenyl cyclase adenyl cyclase. Controlled trials show that both aspirin and ticlopidine are indicated in the secondary prevention of primary prevention. NO, a potent inhibitor of platelets by stimulating guanylate cyclase, elevates cGMP, which, by inhibiting PDE2A, elevates cAMP. By blocking all activating pathways in platelets when cAMP is increased intracellularly, the potential of PDE3 inhibitors is to modulate coronary artery reocclussion. PDE inhibitors have shown promise in animal models and in coronary stents in patients. The immediate goal is to identify critical amino acids in the active site of PDE. This information can be used by pharmaceutical companies to design clinically useful inhibitors with fewer side effects than the current ones. We hypothesize that there are distinct amino acids that facilitate the catalytic reaction, which hydrolyzes cAMP, that form the substrate binding site and that bind essential metal cations. We further postulate that although the inhibitor binding site for cGMP may overlap with the substrate binding site, there are distinct amino acids which interact with this nucleotide. The specific aims of this grant to test this hypothesis are as follows (1) We will synthesize new cGMP-alkylating affinity reagents (8-BDP-TcGMP, 2-BDB-TcGMP, 2-BDB-TeA-3'5'MP and cGMPS-BDB), measure their incorporation, and test their ability to various concentrations to modulate hydrolysis of cAMP and the ability of cAMP, cGMP and AMP to protect against loss of inhibition. (2) We will identify, isolate and sequence the modified peptides labeled by these new cGMP affinity agents and those labeled by the cAMP affinity analog. The function of these residues will be evaluated in the modified enzyme by kinetics, substrate and metal binding. (3) Based on the results of Specific Aim 2, we will design and produce site-directed mutants to test our hypothesis that the amino acids responsible for cGMP binding in the active site of PDE3 differ from those interact with AMP. We will also continue to mutate conserved amino acids and evaluate their effect on enzyme kinetics, Km (cAMP binding), and Ki (cGMP binding), as well as the influence of metals such as Zn++ and Mn++. (4) Since no crystallographic domain based on homologies to other hydrolases in the structural database. A detailed description of PDE3A at the molecular level is important in understanding the role of platelets in hemostasis as well as in the rational design of inhibitors.